This invention relates to the fields of genetic engineering, virus replication, and gene transfer.
Retroviruses and retroviral vectors are potentially powerful tools for the transfer of genes into animals and humans, for the creation of transgenic animals, and for vaccines. However, the use of the retroviruses has certain disadvantages. Most vexatious of them is the obligatory integration of the DNA form of the viral genome for its expression and replication (Cannon et al., 1996; Englund et al., 1995). The integration may have unforeseen and undesired consequences. It could lead to the transcriptional activation through the juxtaposition of the retroviral promoter/enhancer. It could also result in insertional mutagenesis. Both events could result in the production of tumors (Katzir et al., 1985; Neel et al., 1981; Ott et al., 1992), the former by activation of a dominant oncogene, the latter by inactivating a tumor suppressor gene. Other unwanted sequelae may be induced by similar mechanisms (Wu et al., 1993).
Several practitioners in the art have made attempts to circumvent the undesirable consequences of random integration into the host chromosome. For example, they have exploited the idea of tethering the viral integrase protein to the site-specific DNA binding proteins. Such hybrid proteins should recognize specific DNA sequences on the chromosome and integrate the provirus specifically into those regions. However, it is not easy to map all the potential targets in the genome and the resulting hybrid proteins also have not been very efficient (Bushman, 1994; Goulaouic and Chow, 1996). Other strategies, though not entirely successful or simple, were proposed as well.
For example, U.S. Pat. No. 5,118,627 discloses a microbial shuttle vector independently replicative in bacterial and mammalian cells which includes in its DNA sequence bacterial plasmid sequences allowing replication in bacteria, and an SV40 viral ori and SV40 promoters and terminators.
U.S. Pat. No. 5,324,645 discloses a highly retrovirus producing DNA construct having a gene encoding retrovirus, which does not include a retrovirus long terminal repeat sequence. This construct is incorporated into a vector for gene amplification.
U.S. Pat. No. 5,338,674 discloses a live non-pathogenic RNA tumor virus having an altered genome that encodes the antigenic determinants of a pathogen but has no genes coding for pathogenicity.
U.S. Pat. No. 5,420,026 discloses self-assembled, replication defective, hybrid, and virus-like particles having capsid and membrane glycoproteins from at least two different virus types.
Facing the disadvantages associated with retroviral replication involving the obligatory integration step, the instant inventor came forward with a new invention that overcomes these drawbacks. In this application the inventor discloses the construction and testing of novel retroviruses and vectors derived from them that can replicate episomally in vertebrate cells without integration into the chromosomal DNA of the host, therefore rendering them safer for use in gene transfer, in gene therapy, and for vaccines.
This invention is based on a surprising finding that a recombinant hybrid murine leukemia virus (MLV) can replicate in certain mouse cells without integration.
The infectious viral agent such as retrovirus is usually packaged as a virus particle or a virion. It consists of a capsid built of capsid proteins, virus-encoded enzymes, e.g., DNA polymerase, integrase, and protease, a viral RNA genome, which can carry a foreign nucleic acid of interest, and an envelope, in some cases comprising glycoproteins, spanning through the lipid membrane surrounding the particle. Following fusion of the virus envelope with the cellular membrane or endocytosis and virus release into the cytoplasm of the cell, the initial step in the replication involves a reverse transcription step. In this step the RNA genome, which in the case of retroviruses is flanked by short terminal repeats (termed R), is converted to a double-stranded linear DNA form, by virus-encoded reverse transcriptase (RT) or viral DNA polymerase. In this process of reverse transcription, the long terminal repeat (LTR) sequence is produced at each end of the double-stranded DNA. It is this two-LTR configuration that is integrated into the host cell genome by the virus-encoded integrase (IN). Integration occurs at random, or nearly random, sites on host chromosomal DNA. This integrated viral DNA is called a provirus.
Upon retroviral infection almost half of retroviral DNA (Cannon et al., 1996; Leavitt et al., 1996; Yang et al., 1980), goes through an abortive process whereupon the cellular ligase joins both viral ends forming one- or two-LTR circular molecules (Hong et al., 1991; Kulkosky et al., 1990; Pauza, 1990; Smith et al., 1990; Whitcomb and Hughes, 1991; Whitcomb et al., 1990). Under normal circumstances, both one-LTR and two-LTR molecules are rapidly lost from the cell and do not give rise to a new infectious viruses (Englund et al., 1995; Sakai et al., 1993; Stevenson et al., 1990). Thus, the prior art is unambiguous that retroviruses cannot replicate episomally or extrachromosomally unless they integrate into chromosomal DNA of a host cell.
However, this default in retroviral replication, as discovered by this inventor can be overridden. The solution to this problem is originally derived from a replication strategy of a small DNA virus.
One of the best-studied small DNA viruses is the simian virus 40 or SV40 (Shah, 1996). Other small DNA viruses replicate their DNA by a mechanism that closely resembles that of SV40 (Cole, 1996). One skilled in the art would recognize the similarity between the replication strategy described for SV40 and other similar small DNA viruses. For example, bovine papilloma virus or BPV uses so-called E1 and E2 proteins that are similar to analogous counterparts in SV40 (Piirsoo et al., 1996; Lehman and Botchan, 1998).
The instant invention discloses a polynucleotide construct encoding a recombinant retrovirus or vector that is capable, following the conversion of the viral RNA into DNA, of amplification and maintenance of the viral DNA as an extrachromosomal or episomal molecule. As used hereinafter the term episomal replication means that a virus such as retrovirus is able to replicate without integration into host""s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said virus replicates extrachromosomally or episomally. The retrovirus or vector is originally engineered to harbor the sequence coding for the origin of DNA replication or xe2x80x9corixe2x80x9d from a small DNA virus such as SV40. The term origin of DNA replication or ori as used herein means generally a regulatory genetic element found on an episomal DNA molecule allowing the replication of said DNA molecule so that these DNA molecules are not eventually lost upon division of a host cell. This mechanism assures stable extrachromosomal or episomal replication. Following infection, the double-stranded retroviral DNA is generated by means of reverse transcription and circularized upon entry into the host cell. The retrovirus carries ori and the host cell provides the cognate replication protein of small DNA virus, which is used for amplification and replication of the circular DNA retroviral genome. A replication protein can be so-called large T antigen of SV40 or it can be E1 or E2 of BPV. This is the first known hybrid retrovirus that does not have to integrate into the host genome in order to replicate efficiently in the host cells.
The instant invention also discloses a recombinant retrovirus or vector that is capable of amplification and maintenance of the viral DNA as an extrachromosomal molecule with the aid of minichromosome maintenance element (MME)xe2x80x94a regulatory element from bovine papilloma virus (BPV), which allows stable replication of episomal constructs without gradual loss from the host cell (Piirsoo et al., 1996).
Furthermore, the instant invention also discloses a recombinant retrovirus or vector that is capable of amplification and maintenance of the viral DNA as an extrachromosomal molecule without aid of ori and replication protein of the host cell. This is the first known hybrid retrovirus that does not integrate into the host genome in order to replicate efficiently in the host cells and does not use extraneous replication proteins.
The hybrid retroviruses described above are the first examples of a retrovirus that does not integrate into the host genome and therefore represents a novel and very important discovery that attains set forth goals of safety for medical purposes. Thus, the most important feature of this invention is the discovery that a recombinant retrovirus can efficiently replicate in host, e.g., human cells without integration. This feature is accomplished by using various recombinant strategies such as use of ori or MME and related elements, amphotrophic envelope, allowing stable episomal replication without loss from the host cell. Another important feature is a recombinant virus or vector replicating episomally without extraneous regulatory elements like ori or MME and replication proteins like large T antigen or E1 and E2.
The present invention provides a recombinant virus, viral vector, or transposon that has a novel replication strategy and which allows the creation of a virus that would replicate in vertebrate cells via reverse transcription without the danger of being xe2x80x9csilencedxe2x80x9d or of deregulating host genes upon integration into the host chromosome. The term transposon as used herein means a type of transposable element which, in addition to genes involved in transposition, carries other genes: often conferring selectable phenotypes such as antibiotic resistance or ability to replicate under unfavorable conditions. The term transposition as used herein means the movement of a fragment of nucleic acid around the template, usually through the function of transposable element. The term transposable element as used herein means a genetic element that has the ability to move (transpose) from one site on a template to another. The yeast Ty1 is a good example of LTR retrotransposon family.
In particular, the origin of DNA replication derived from a DNA virus is inserted into the genome of a virus that undergoes reverse transcription as a regular part of retroviral life cycle. After reverse transcription of the viral genomic RNA into DNA, the resulting molecule is circularized by cellular enzymes and, subsequently, replicates further as extrachromosomal DNA. Such extrachromosomal replication results in the amplification of this DNA intermediate in the cell, which, in turn, allows for viral replication and the production of either high titer viral stock or replication defective vectors. This process provides viruses and their respective vectors, which are quite safe for therapeutic purposes, such as gene transfer/therapy and other medical applications, such as viral or cancer vaccines. The said vaccines comprise both prophylactic vaccines and therapeutic vaccines.
Accordingly, it is a general object of the present invention to provide recombinant viruses, transposons and their vectors, which are capable, following the conversion of retroviral RNA into DNA, of amplification of same in the cell as episomes via DNA replication. The replication may be by any means except as a result of integration into the host chromosome, including, but not limited to, the presence of DNA sequences from DNA viruses, which define an ori or MME and encode a DNA replication protein. The replication may be by any means except as a result of integration into the host chromosome, without native DNA sequences from DNA, viruses, which define an ori or MME and encode a DNA replication protein such as T antigen or E1 and E2. The resultant recombinant viruses replicate without the necessity of integration into the host chromosome. The resulting virus can be infectious or non-infectious. The determination of that depends on viral myristilation of the gag protein, by differential splicing, by differential initiation of RNA transcription, and by the environment of the host cell or organism. The term non-infectious virus as used herein also means non-pathogenic virus, while infectious virus can be both pathogenic and non-pathogenic.
It is a further object of the present invention to provide an episomally replicating polynucleotide construct comprising retroviral sequences encoding at least one LTR of a retioviral genome, said retroviral sequences further comprising one or more mutations that disable the integration of said construct into host chromosomal DNA, said construct further having the capacity to replicate via reverse transcription, provided that any reverse transcription product obtained from such reverse transcription is also disabled from integrating into host chromosomal DNA.
It is a further object of the present invention to provide an recombinant temperate viruses, transposons and their vectors that are capable of infecting non-dividing cells.
It is a further object of the present invention to provide recombinant temperate viruses, transposons and their vectors that are capable of replicating in synchrony with host""s replication cycle without causing cell lysis.
It is a further object of the present invention to provide recombinant viruses, transposons and their vectors that are capable of replicating via a xe2x80x9clyticxe2x80x9d cycle, which would kill the host cell.
It is a further object of the present invention to provide recombinant viruses, transposons and their vectors that are capable of replication only in certain specific cells including but not limited to cells already infected with DNA virus which would provide necessary elements.
It is a further object of the present invention to provide a recombinant viruses, transposons and their vectors that are capable of replication only in certain specific cells, including but not limited to, tumor cells already infected with DNA virus, and capable of killing these cells in vitro and in vivo by means of, including but not limited to, run away replication, or by a toxic gene expression.
It is a further object of the present invention to provide a recombinant viruses, transposons and their vectors that have altered integrase protein or protein function and/or the cis elements necessary for integration or virtually any part of the genome that enhances the circularization of viral DNA and attenuates or abolishes integration.
It is a further object of the present invention to provide recombinant viruses, transposons and their vectors that have altered integrase protein or protein function but are able to replicate extrachromosomally and indefinitely without aid of SV40 T antigen or any extraneous native ori element or MME-like element from a small DNA virus.
It is a further object of the present invention to provide a recombinant viruses, transposons and their vectors which are either replication competent or defective that are capable of carrying a nucleic acid sequences of interest, infect host cells and express such a sequence in vertebrates or their cells in culture.
It is a further object of the present invention to provide a recombinant viruses, transposons and their vectors that are capable of carrying a nucleic acid sequences under the control of late promoters, such as SV40 late promoter or poxvirus late promoter, whose products are toxic to cells and may or may not alter or kill the cells.
It is a further object of the present invention to provide a recombinant viruses, transposons and their vectors that are capable of carrying a nucleic acid sequences devoid of the control of late promoters, such as SV40 late promoter or poxvirus late promoter, whose products are toxic to cells and may or may not alter or kill the cells.
It is a further object of the present invention to provide a recombinant viruses whose genomes are derived from but not limited to human immunodeficiency virus (HIV), Human T Lymphotropic Virus type I or II (HTLV), simian immunodeficiency virus (SIV), simian sarcoma virus (SSV), Rous sarcoma virus (RSV), caprine arthritis-encephalitis virus (CAEV), murine leukemia virus (MLV), avian leukemia virus (ALV), bovine leukemia virus (BLV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EAIV), and endogenous retroviruses (ERV), including human endogenous retroviruses (HERV) which would use the replication strategy of this invention.
It is a further object of the present invention to provide host cells carrying the viruses, transposons and their vectors capable of expressing one or more nucleic acid sequences or genes and supporting the replication of such sequences or genes.
It is a further object of the present invention to provide tissue culture cells allowing generation of high titer virus stock by growing the cells transfected with the viral construct and allowing the virus to spread.
It is a further object of the present invention to provide a method for selection of viruses, transposons and their vectors for better replication including the use of marker gene transfer.
It is also an object of the present invention to provide methods for viruses, transposons and their vectors to transfer nucleic acid sequences into broad range of organisms or host cells of vertebrates, particularly humans both in vitro and in vivo.
It is also an object of the present invention to provide a means of generating transgenic animals.
It is a further object of the present invention to provide transgenic animals carrying the recombinant virus in at least one cell and capable of supporting the replication of thereof.
It is a further object of the present invention to provide methods for preventing and treating various diseases by gene transfer and vaccination and transfer of virus or vector harboring a nucleic acid sequence of interest to vertebrate hosts, preferably humans.
It is a further object of the present invention to provide methods for diagnostics of various diseases including but not limited to tumor cells or cells infected with other viruses.
These and other objects of the invention will become apparent by reference to certain examples, which are included herein for purpose of illustration and are not intended to be limiting.